Many bacteria and higher plants produce cytotoxic proteins collectively called ribotoxins which function by being taken up by, and then inactivating the ribosomes of a target cell. The ribotoxins are considered to fall into two major classes: NAD.sup.+ -dependent ribotoxins, which appear to disable ribosomes by covalently attaching ADP-ribose to "elongation factor-2" protein, and NAD.sup.+ -independent ribotoxins, which appear to inactivate the 60S ribosomal subunit. It is the NAD.sup.+ -independent ribotoxins and their derivatives to which the separation and purification methods of the invention apply. These ribotoxins affect only eucaryotic ribosomes and because they function enzymatically, they are lethal at low concentrations.
Some of the ribotoxins with which the invention is concerned can be isolated as covalently linked heterodimers consisting of an enzymatically active (ribotoxic) A chain polypeptide linked through a disulfide bond to an enzymatically inactive B chain polypeptide which is responsible for binding to the target cell surface, and which may also facilitate uptake of the cytotoxic portion. Representative heterodimeric ribotoxins which are subject to the methods of the invention are ricin, abrin, and modeccin.
Other ribotoxins are single polypeptides which are cytoloxioally active, and are thus sometimes referred to as "A chain toxins" or "hemitoxins". Examples of such A chain toxins to which the method of the invention is applicable are pokeweed antiviral protein (PAP), mitogillin, restrictosin, momordin, saponarin, and gelonin.
Several ribotoxins, such as ricin, abrin, and PAP, occur in nature in more than one form. Thus, these ribotoxins can be considered to represent several isotoxins--i.e. structurally similar proteins with quantitatively differing functional properties.
Recent reviews of ribotoxins in general include Olsnes, et al, Molecular Action of Toxins and Virus, Cohen, et al, ed (1982) E1 sevier, N.Y. pp 51-105; and Barbieri, et al, Cancer Surveys (1982) 1:488-520.
Some attempts have been made to take advantage of the cytotoxic properties of the ribotoxins by employing the unmodified polypeptides as therapeutic agents (see, for example, Fodstad et al, Cancer Research (1984) 44:862-862). However most efforts to use ribotoxins therapeutically have been focused on hybrid toxins, in which the cytotoxic moiety is covalently coupled to a "binding moiety" expected to bind specifically to certain cells, virus, or other macromolecules. The most common examples of hybrid toxins are immunotoxins, wherein the cytotoxic polypeptide is conjugated to a specific antibody; however, a variety of other binding moieties may be used.
Many examples of the utility of such hybrid toxins are available. In the simplest concept, an antibody is chosen to recognize an antigen characteristic of an undesired target cell such as a cancer cell. The antibody seeks out the target cell and permits the toxin to kill it. In slightly more complex applications, a conjugate between the toxic polypeptide and an antigen recognized by autoimmune lymphocytes responsible for destroying normal tissue is administered to patients with autoimmune diseases. Hybrid toxins may also be used to aid in preventing transplant rejection either by destroying the lymphocyte cells attacking the foreign material, or by eliminating undesirable cells, accompanying the transplant, which attack host cells.
The hybrid toxins are prepared by covalent chemical coupling of a purified ribotoxin to a binding moiety and purification of the resulting conjugate by separation from unconjugated components. The ribotoxin component may itself need to be generated by reduction of a heterodimeric ribotoxin and separation of the resulting A and B chains from the unreacted heterodimer. The methods of purification provided by the invention are useful in obtaining pure whole ribotoxin, in obtaining pure ribotoxin A chain from heterodimer and in purifying the hybrid toxins from the conjugation mixture.
Hybrid toxin preparation and therapeutic effect have been reviewed by Thorpe, et al, Immunol Revs (1982) 62:119-157 and by Edwards, Pharm Ther (1983) 23:147-177. Ricin, abrin, their purified A chains, PAP, gelonin, and diphtheria toxin A chain have, for example, all been employed in hybrid toxin preparation and use. When, as usually is the case, hybrid toxins include only the A chains of heterodimeric ribotoxins in order to minimize non-specific binding, the putative advantageous property of the B chains to mediate translocation of the cytotoxic portion into the cell is lost. Optimization of hybrid toxin performance may be possible by selecting whole toxins for conjugation which have desirable intrinsic binding specificities.
Purification steps are critical in preparing any material for therapeutic use. In the particular case of ribotoxin hybrid toxins the preparation just not only be free of impurities from original extracts, but also from unreacted components and side products of the conjugation reaction, and specifically from unwanted B chain moieties derived from a heterodimer. In some applications, homogeneity with respect to the stoichiometric ratio of cytotoxin to binding moiety ratio (i.e. the multilicity) may be beneficial. Generally, conjugation procedures result in mixtures wherein the antibody, for example, is conjugated to 1, 2, 3, or more cytotoxic moieties. Such heterogeneity may complicate the interpretation of results obtained with the preparation, and may affect the therapeutic value as well.
In addition effective purification of the ribotoxin moiety prior to conjugation is more important than it might at first appear. For example, the additional oligosaccharide chain on RTA2 (one of two isoenzyme A chains derived from ricin; see below) may be recognized by the mannose/N-acetylglucosamine clearance system (Stahl, et al, Proc Natl Acad Sci (U.S.A.) (1978) 75:1399-1403; Kawasaki, et al, Biochem Biophys Res Commun (1978) 81:1018-1024). Therefore, conjugates formed with RTA2 may be cleared from circulation more quickly than those formed with the other isoenzyme, RTA1; if so, therapeutic efficacy may be reduced. Ideal hybrid toxins would be formed from purified RTA1 which is, putatively, less rapidly cleared from the blood.
The invention herein provides a successful method to obtain homogeneous preparations of hybrid toxins. It permits separation of hybrid toxins of varying stoichiometry, and also permits separation of isotoxins both of full heterodimeric polypeptides and of ribotoxin A chain. In addition the invention provides a useful method for purifying ribotoxins from their natural sources.
By application of the method of the invention, a previously unknown isotoxin ricin E2, which has unique properties differing from those of the previously known ricin D and ricin E, has been isolated and characterized. Ricin E2 has cytotoxicity levels and binding specificities which result in hybrid toxin derivatives having superior ratios of target-specific cytotoxcity to whole-animal toxicity as compared to corresponding RTA hybrid toxins.